Different properties of blood provide information on the condition of the blood's provider. For example, anemia is a reduction in total circulating red blood cell mass, diagnosed by a decrease in hemoglobin concentration. In addition, a variety of detrimental red blood cell conditions such as hereditary spherocytosis and thalassemia are indicated by fragile blood cell membranes. Measuring these properties has historically taken relatively large samples of blood and time. This invention reduces both the sample size and the time for property determination.
According to E. Uthman, Understanding Anemia, University of Mississippi Press, Chapter 1, 1998, red blood cells (RBCs) are oxygen transporters. They are basically membrane structure filled with hemoglobin (a 33% solution). This pigmented protein accounts for both their natural red color and affinity for acidic dyes, such as eosin. They retain a cytoskeleton which gives them distinctive biconcave architecture. RBCs are also highly flexible and resilient, which is important for passage through capillary lumens. The shape of the red cell is referred to as a biconcave disc. (A donut with its hole partially filled in is a good analogy.) Red blood cells are about 7.5 .mu.m in diameter and 2-3 .mu.m in thickness, but they must be pliable enough to squeeze through smaller capillaries at high velocity. The critical importance of cytoarchitecture is highlighted by diseases such as sickle cell anemia in which RBC shape is distorted, resulting in blocked capillaries and frequently breakage (hemolysis) of the fragile sickle shaped cells.
Estimates suggest that 20% of the world's population suffers from various anemias arising from dietary or genetic deficiencies. Anemic patients have low oxygen-carrying capacity of the blood, with resultant tissue hypoxia. The clinical symptoms are related to the severity of the anemia, and may include pallor, tachycardia, angina, light-headedness and fatigue. Anemia may be due to increased blood loss, decreased red blood cell production (hypoproliferative anemia), or increased red blood cell destruction (hyperproliferative anemia).
Hemoglobin is a protein that serves as a carrier for oxygen from the lungs to the tissues. To work properly, the hemoglobin has to hold on to oxygen molecules with just the right amount of force. If the hemoglobin molecule binds the oxygen molecules too loosely, then it will not be capable of picking them up at the lungs. If it binds the oxygen too tightly, then when it gets out to the tissues it will not release the oxygen to the tissues that need it. Hemoglobin has to have its peculiar structure for proper oxygen transport, even if that structure turns out to be very delicate. Almost any type of natural or artificial toxic substance can cause the hemoglobin molecule to denature (be permanently altered so that it does not work).
According to the Merck Manual, 16.sup.th ed, 1992, anemia results from one or more combinations of 3 basic mechanisms: blood loss, decreased RBC production, or increased RBC destruction (hemolysis). For diagnosis of anemia, once blood loss is ruled out, only the other 2 mechanisms remain. Since RBC survival is 120 days, maintenance of steady populations requires renewal of 1/120 of the cells each day. Complete cessation of RBC production results in a decline of about 10%/wk (1%/day) of their initial number. When RBC values fall&gt;10%/wk (ie, 500,000 RBC/.mu.L) without blood loss, hemolysis is established as a causative factor.
The presence of anemia is typically detected with a blood count that is historically made by mixing a measured volume of blood with an appropriate diluent or lysing agent and counting RBCs in a chamber under the microscope. There have been many systems deviced to automate this procedure.
U.S. Pat. No. 4,199,748 of J. Bacus discloses a system for diagnosing anemia by imaging a number of cells and determining characteristics based on cell shape.
U.S. Pat. No. 5,194,909 of D. Tycko discloses a system for measuring hemoglobin concentration from a stream of individual, spaced-apart cells passing through a flow chamber.
U.S. Pat. No. 5,686,309 of R. Frank et al. discloses a system for determining hemoglobin content by measuring the electrical parameters of individual red blood cells.
U.S. Pat. No. 5,793,485 of P. Gourley, Resonant-Cavity Apparatus for Cytometry or Particle Analysis, discloses a resonant-cavity apparatus for analyzing cells which includes a cavity formed by at least two spaced reflecting mirrors that contains a semiconductor gain medium and an analysis region for containing one or more cells or particles to be analyzed. At col. 17, lines 1-30, the patent discusses how the emission spectrum of the light beam through the cavity can be used to recover information about the size and shape of a cell or particle, or components of the cell or particle, in the cavity. However, there is no teaching in this earlier work of this invention.
According to Ian Russell, Delmar's Clinical Laboratory Manual Series: Hematology, Delmar Publishers, New York 1997, most routine blood cell analysis such as blood counts are carried out with automated instrumentation. Accordingly, many modern laboratories do not prepare manual differential slides on all blood samples. However, manual differential provides information that a computerized count could not, such as specific red cell morphology, platelet morphology, and presence of inclusion bodies. When any results are abnormal in the automated cell count, a differential slide is often made. However, this process is labor intensive and time consuming.
An important feature of red blood cells is osmosis and membrane permeability, the ability of membranes to selectively transport fluids. The cells are freely permeable to water and anions like Cl.sup.- and HCO.sub.3.sup.- but nearly impermeable to cations like Na.sup.+ and K.sup.+, so the cell maintain volumes and homeostasis. Integral membrane pumps driven by Adenine Triphosphate (ADT) transport Na.sup.+ out and K.sup.+ into the cell, respectively.
If red blood cells are placed in an isotonic (therefore isosmotic) solution, they will be in osmotic equilibrium and the cells will neither swell and burst (lyse) nor shrink (crenate). However if the cell membrane is permeable to the solute and initially there is a decreasing concentration gradient from outside to inside the cell, solute will diffuse into the cell and add to the solute already present there. Eventually the osmotic potential of the cytosol will be greater than the osmotic potential outside and the bathing solution will have become hypotonic. The natural consequence is for osmosis to occur, for water to diffuse into the cell, producing lysis. In lysing red blood cells, the biconcave shape changes to spherical and the membrane ruptures. The time it takes for lysis to occur is an indirect function of permeation rate. The physical rupture of the bilipid membrane under tension has been analyzed theoretically and found to be a thermally activated process.
The biocavity laser of this invention can eliminate much expensive testing by providing a more thorough analysis of blood cell morphology, especially for the determination of cell size, shape, and distribution in a large population.